Pref-1 (preadipocyte factor-1) is a novel plasma membrane protein containing six EGF-like repeats in the extracellular domain. Pref-1 is highly expressed in 3T3-L1 preadipocytes, but is not detectable in mature fat cells. Constitutive expression of pref-1 by transfection inhibits and antisense oliogonuceotide treatment enhances adipocyte differentiation, demonstrating inhibitory role of pref-1 in adipogenesis. We also found that pref-1 ectodomain is cleaved to generate a biologically active 50 kD soluble form. Unlike classic growth factors that generally inhibit differentiation by preventing cell cycle withdrawal that must precede the differentiation process, pref-1 does not have mitogenic effects. pref-1, made and secreted by preadipocytes, may be a molecular checkpoint early in adipogenesis that keeps cells in an undifferentiated state. The goal of this research is to elucidate the mechanisms underlying the antiadipogenic action of pref-1 and the mechanisms for pref-1 downregulation during adipoconversion. Aim 1 is to further characterize pref-1 in vitro for its function and structural requirements in inhibiton of adipocyte differentiation. We will address whether membrane-anchored pref-1 is biologically active as is the soluble form. The potential additional function of cytoplasmic domain, and the structure and processing sites for the soluble form will be examined. We will also identify the minimal pref-1 ectodomain necessary for its antiadipogenic action. Aim 2 is to clone the pref-1 receptor that mediates pref-1 action and signaling. We are employing mammalian expression cloning using pref-1-alkaline phosphatase fusion protein as ligand. Identification of the pref-1 receptor will give us key insights to pref-1 action. Aim 3 is to further examine the role of pref-1 by creating transgenic mice ectopically expressing pref-1 in a tetracylcine regulated manner and in the pref-1 knockout mice we have generated. Aim 4 is to clone the transcription factor that binds to the negative element we recently defined to be responsible for differentiation-dependent downregulation of the pref-1 gene in adipocytes. This research is directed toward understanding adipocyte differentiation, a critical process that contributes to the development of excess adipose mass and, therefore, obesity. In the long run, elucidating the mechanisms of pref-1 function and regulation will allow us to begin to develop strategies to control adipogenesis by modifying pref-1 action and by altering expression of preadipocyte genes including pref-1, that control adipogenesis.